The analysis of the human genome is expected to be completed in the early twenty-first century. For effective utilization of the outcome of the analysis, it is indispensable to develop a new technology for artificial manipulation of nucleic acids (carrying, sequence recognition, and control of transcription or translation of nucleic acids). The most important material for manipulating nucleic acids is considered to be a carrier capable of interacting with a nucleic acid such as DNA to support or carry the nucleic acid. However, in-vivo uses of conventional gene carriers composed of artificial materials have produced no significant results in human clinical studies. This can be attributed especially to (1) low gene-transferring efficiency, (2) difficulty in controlling the association and dissociation of genes (Cotton et al., Meth. Enzymol. 217: 618-644 (1993)); and (3) cytotoxity caused by cationic carrier materials (Choksakulnimitr et al., J. Control. Rel., 34: 233-241 (1995)).
Although viruses such as retroviruses (Miller, Nature 357: 455-460 (1992)) or adenoviruses (Mulligan, Science 260: 926-932 (1993)) have shown very promising in-vitro results as gene carriers, in-vivo use of these naturally occurring materials is restricted, especially because of inflammatory action, immunogenetic properties or the risk of integration into the genome or mutagenesis induction due to the viruses (Crystal, Science 270: 404-410 (1995)). Thus, as a substitute for such naturally-originating gene vectors, there has been proposed use of non-viral carriers composed of an artificial material which can be handled in an easier manner and can carry DNAs into the cells in a more efficient manner as compared with the viruses (Tomlinson and Rolland, J. Contr. Rel., 39: 357-372 (1996)).
At present, it is polyethyleneimine (PEI) that is the most extensively studied non-viral, artificial carrier material. It has been shown that PEI, a cationic polymer assuming a three dimensional branched structure, may result in transfection in a considerably highly efficient manner for various adhesive and floating cells, (Boussif et al., Gene Therapy 3: 1074-1080 (1996)). For example, 95% in-vitro transfection was accomplished in the 3T3 fibroblast cell line. In-vivo gene transfection into mouse brain using PEI as a carrier resulted in long-term expressions of the reporter gene and Bcl 2 gene in the neuron and the glial cell, the results being comparable to those obtained with the gene transfection using the adenovirus (Abdallah et al., Hum. Gene Ther. 7: 1947-1954 (1996)).
However, the safety of cationic polymers such as polyethylimine has not yet been established. While the introduction of amino groups is indispensable for imparting a cationic charge to such a polymer, an amino group has a risk of toxicity in the living body due to its high physiological activity. In fact, no cationic polymers studied so far have yet been put into practice, or yet been registered in the “Pharmaceutical Additives Handbook” (edited by the Pharmaceutical Additives Association of Japan and published by Yakujinipposha Publishing Co.).
β-1,3-glucan is a polysaccharide which has been clinically put to practical use as an intramuscular injection. It has been known since a long time ago that the polysaccharide takes, as it occurs naturally, a triple-stranded helix structure (cf., for example, Theresa M. McIntire, David A. Brant, J. Am. Chem. Soc., Vol. 120, 6909 (1998)). This polysaccharide has already been confirmed with respect to its in-vivo safety, based on the results of practical use for some 20 years as an intramuscular injection (Simizu et al., Biotherapy, Vol. 4, 1390 (1990); Hasegawa, Oncology and Chemotherapy, Vol. 8, 225 (1992)).
PCT/US95/14800 teaches that β-1,3-glucan is chemically modified so as to be capable of forming a conjugate with a bioactive material such as DNA, for use as a gene carrier. However, this prior art describes nothing but a preparation of a β-1,3-glucan/bioactive material conjugate wherein the β-1,3-glucan is utilized as it occurs naturally, i.e. in the form of a triple-stranded helix structure, to form the conjugate through covalent coupling.
Recently, the present inventors and others have found that β-1,3-glucan can form, as it is artificially treated, a new type of complex with a nucleic acid (PCT/JP00/07875; Sakurai and Shinkai, J. Am. Chem. Soc., 122, 4520 (2000); Kimura, Koumoto, Sakurai and Shinkai, Chem. Lett., 1242 (2000)). More specifically, it has been found that the triple-stranded helix form, which the polysaccharide takes as it naturally occurs, can be unbound into separate single strands by dissolving the triple helix (triple-stranded helix) polysaccharide in a polar solvent. Thus, as the solution is added with a single-stranded nucleic acid and the solvent is replaced by water (as a renaturation process), there is formed a triple-stranded helix complex composed of a single-stranded nucleic acid and a double-stranded polysaccharide. The binding between the polysaccharide and the nucleic acid in the complex is considered to be primarily through hydrogen bonding. (K. Sakurai, R. Iguchi, T. Kimura, K. Koumoto, M. Mizu, and S. Shinkai, Polym. Preprints, Jpn., 49, 4054 (2000)). The energy of this binding is relatively small, resulting in an easy dissociation of the complex. It is necessary to render this complex more strongly affinitive with nucleic acids in order to utilize the complex as a gene carrier.
It is an object of the present invention to provide a new type of gene carrier which is capable of interacting with and bonding to a nucleic acid such as DNA and RNA, without destroying the nucleic acid, to form a water-soluble complex so as to be applicable under biological conditions, and which is also capable of dissociating the nucleic acid from the complex and rebonding to such nucleic acid when necessary.